The long-term objectives of the proposed research are to identify factors that play important roles in transcription by RNA polymerase II in vivo and to determine the mechanisms by which these factors govern this process. The central focus of this grant is the TATA box-binding protein (TBP), which plays an essential role in transcription initiation by all three nuclear RNA polymerases. At promoters transcribed by Pol II, binding of TBP to the TATA box is the first step in a cascade of events that culminates in the production of an RNA message. Therefore, an understanding of the factors that control TBP is a critical step toward understanding the regulation of gene expression in eukaryotes. The Specific Aims represent extensions of previous studies on Saccharomyces cerevisiae TBP. Specific Aim 1 is to investigate the function of RTF1, a protein that is important both for TATA site selection by TBP and for transcript elongation. RTF1 and associated proteins will be purified, RTF1-regulated genes will be identified using DNA microarrays, and genetic selections will be performed to identify extragenic suppressors of rtf1 mutations. In addition, the phosphorylation state of Pol II, which correlates with the transcription cycle, will be analyzed in rtf1 and other mutant strains. Specific Aim 2 is to elucidate the importance of TBP as a target for transcriptional regulatory factors in vivo. Chromatin immunoprecipitation and in vivo footprinting methods will be used to determine the effect of gene-specific and globally acting transcription factors on the recruitment of TBP to a highly regulated model promoter. Specific Aim 3 is to analyze two distinct classes of TBP mutants and gain insights into two fundamental aspects of Pol II transcription: orientation specific assembly of the preinitiation complex and regulation of transcription initiation. TBP mutants that exhibit reversed DNA binding polarity in vivo will be analyzed using in vitro DNA cleavage and transcription assays. TBP mutants that are altered in a particular subdomain of TBP and exhibit novel phenotypes, indicative of defects in transcriptional regulation, will be studied using both biochemical and genetic approaches. Since the proteins and mechanisms employed in the regulation of RNA polymerase II are conserved throughout eukaryotes, the information that is learned from these studies in yeast will significantly advance our understanding of transcription in humans, where a disruption in this process leads to important human diseases including cancer and AIDS.